Method of producing transgenic maize using direct transformation of commercially important genotypes

ABSTRACT

Methods for transformation of maize with nucleic acid sequences of interest are disclosed. The method involves subjecting immature zygotic embryos or Type I callus to high velocity microprojectile bombardment. The method is capable of producing transformed maize lines of commercial importance and their hybrid combinations.

This application is a continuation-in-part application of U.S. Ser. No.951, 715 filed Sep. 25, 1992, which is a continuation-in-partapplication of U.S. Ser. No. 772, 027 filed Oct. 4, 1991 whichdisclosures are herein incorporated in their entirety.

FIELD OF THE INVENTION

The present invention relates to the transformation of maize genotypesby microprojectile bombardment.

BACKGROUND OF THE INVENTION

The use of genetic engineering to introduce new agronomically importanttraits into maize such as insect resistance will have many commercialbenefits. In order to accomplish this in the most expedient fashion itis necessary to have a method of transformation that can be used withmaize genotypes that are commercially valuable.

The majority of instances of maize transformation have used a genotypeknown as A188, or derivatives of A188. This is because these lines areeasily established in vitro as an embryogenic line that forms Type II,or friable, embryogenic callus and suspension cultures. Such Type IIcultures have been exclusively preferred as a recipient of introducedgenes. in transformation methods. Unfortunately, A188 is an inferiorinbred for the development of commercially important hybrids. (Hodges etal., Biotechnology, 4:219, 1986). Working with such “model” maize linesas A188 is disadvantageous in that extensive breeding is usuallyrequired in order to develop maize lines with a desirable geneticcomposition. What is needed is a method that can be used withcommercially valuable maize lines without the need for reliance on such“model” systems based on Type II or suspension cultures.

Microprojectile bombardment has been advanced as an effectivetransformation technique for cells, including cells of plants. InSanford et al., Particulate Science and Technology, 5: 27-37 (1987) itwas reported that microprojectile bombardment was effective to delivernucleic acid into the cytoplasm of plant cells of Allium cepa (onion).Christou et al., Plant Physioloqy 87: 671-674 (1988) reported the stabletransformation of soybean callus with a kanamycin resistance gene viamicroprojectile bombardment. Christou et al. reported penetration atapproximately 0.1 to 5% of cells. Chri.stou further reported observablelevels of NPTII enzyme activity and resistance in the transformed calliof up to 400 mg/L of kanamycin. McCabe et al., Bio/Technology 6: 923-926(1988) report the stable transformation of Glycine max (soybean) usingmicroprojectile bombardment. McCabe et al. further report the recoveryof a transformed R₁ plant from an R₀ chimeric plant.

Transformation of monocots and, in particular, commercially. valuablemaize lines, has been problematic. Although there have been severalreports of stable plant transformation utilizing the microprojectilebombardment technique, such transformation has not resulted in theproduction of fertile, regenerated transgenic maize plants of acommercially. valuable genotype—each report used the genotype A188 orits derivatives (Fromm et al, BioTechnology, 8:833, 1990; Walters etal., Pl. Mol. Biol. 18:189, 1.992, Gordon-Kamm et al., Plant Cell,2:603, 1990). There are two reports of maize transformation usingcommercially valuable lines but both rely on the availability of TypeII, friable embryogenic callus as a recipient for gene delivery (Jayneet al., 1991 Meeting of the International Society for Plant MolecularBiology, Abstract #338; Aves et al., 1992 World Congress on Cell andTissue Culture, In Vitro 28:124A, Abstract #P-1134).

Prior to the present invention, successful direct transformation ofcommercially valuable maize lines has not been achieved usingmicroprojectile bombardment of immature zygotic embryos or Type Iembryogenic callus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the plasmid map of the vector pCIB3064 containing the35S/bar chimeric gene.

FIG. 2 shows the plasmid map of the vector pCIB3089 containing the35S/B-Peru chimeric gene.

FIG. 3 shows the plasmid map of the vector pCIB4421 containing the PEPCarboxylase promoter fused to the synthetic BT coding sequence.

FIG. 4 shows the plasmid map of the vector pCIB4430 containing thepollen-specific promoter fused to the synthetic BT coding sequence.

FIG. 5 shows the plasmid map of the vector pCIB4431 containing thepollen-specifc promoter fused to the synthetic BT coding sequence andthe PEP Carboxylase promoter fused to the synthetic BT coding sequence.

FIG. 6 shows the plasmid map of the vector pCIB4433 containing thepith-preferred promoter fused to the synthetic BT coding sequence andthe 35S/bar chimeric gene.

FIG. 7 shows the plasmid map of the vector pCIB4436 containing the35S/C1 chimeric gene.

SUMMARY OF THE INVENTION

The present invention is drawn to the stable transformation of maizewith nucleic acid sequences of interest, the regeneration of fertiletransgenic maize plants and their subsequent use for the creation ofcommercially valuable lines and hybrids created with those lines. In theinvention, immature zygotic embryos are subjected to high velocitymicroprojectile bombardment as a means of gene delivery within about 14days after excision from the plant. After initiation of embryogeniccallus and selection for transformed cells, stably transformed plantsmay be regenerated which express the foreign genes of interest.Alternatively, callus derived from immature zygotic embryos having theType I character may also be employed as a recipient for gene delivery.The method is applicable to any genotype of maize, especiallycommercially important ones. In this manner, the method producestransformed maize lines of commercial importance and their hybridcombinations.

DETAILED DESCRIPTION OF THE INVENTION

A method for the transformation of any maize line and the regenerationof transgenic maize plants is provided. The method involves the deliveryof nucleic acids, or particularly, genes of interest, directly toimmature zygotic embryos. Alternatively, said nucleic acids or genes ofinterest may be delivered to serially-propagated Type I embryogeniccallus obtained from immature zygotic embryos. Stably transformed cellsare obtained and are regenerated into whole, fertile plants thatexpress-the foreign gene(s). Furthermore, the fertile transformed plantsare capable of producing transformed progeny that express the foreigngene(s). The method provides for the direct transformation ofcommercially important maize genotypes, for obtaining transformedinbreds and for their hybrid combinations.

Embryogenic callus of maize is obtained by the process of somaticembryogenesis. Somatic embryogenesis is a process by which fully orpartially-formed embryos arise from somatic tissue. This- is in contrastto zygotic embryogenesis whereby embryos form from gametic tissue.Somatic embryogenesis may -be induced from several viable tissues ofmaize, including leaf bases (Conger et al., Pl. Cell Rep. 6:345, 1987),tassel primordia (Rhodes et al., Pl. Sci. 46:225, 1986) and immatureembryos (Green et al., Crop Science 15:417, 1975). In the presentinvention, immature embryos are the preferred source of embryogeniccallus.

Immature zygotic embryos of maize can be obtained by pollinating an earwith viable pollen then removing the ear from the plant at some latertime. Typically, immature embryos for use as sources of embryogeniccallus are in the size range of about 0.5 mm to about 3.0 mm, moreparticularly about 1.0 to about 2.5, especially preferred from 1.5 to2.0. The immature embryos may, for example, be removed from the earindividually by dissection or in bulk by “creaming” the kernels.Isolated immature embryos are placed with the zygotic embryo axis sidein contact with an appropriate nutrient medium (Green et al., supra,1975). Embryogenic callus is typically observed on the immature embryowithin about 14 days of placement on the medium. This initial period iscalled the “initiation” step. After the initiation step, the embryogeniccallus is generally transferred to a different medium for establishmentand maintenance of serially-propagatable embryogenic callus (called the“maintenance” medium), although the same medium as that for “initiation”may also be used. Incubation of cultures typically takes place at 25 C.in the dark or under low light. Embryogenic callus can be obtained inthis way from a wide variety of maize genotypes.

Two main types of embryogenic callus have been described in thescientific literature. Type I embryogenic callus has been defined as“translucent, convoluted and compact callus” (Tomes et al., Theor. Appl.Genet. 70:505-509, 1985) or as “compact, morphologically complex”(Armstrong et al., Planta, 164:207-214 (1985)). Type II embryogeniccallus has been defined as “friable and fast growing [callus] with welldefined somatic embryos with suspensor-like structures” (Tomes et al;,supra, 1985) or as “friable, embryogenic” (Armstrong et al., supra,1985). It is within the scope of this invention that either or bothtypes of embryogenic callus may be obtained from the immature zygoticembryos, or other viable tissue such as tassel primordia, leaf bases ormeristems which may also be used as a source of embryogenic callus.

In order to obtain embryogenic callus the isolated immature maizeembryos must be cultured on an appropriate medium. Many types of mediumhave been shown to be useful for the establishment of embryogenic callusfrom a variety of genotypes, including-some commercially important ones(Hodges et al., Bio/Technology 4:219, 1986; Duncan et al., Planta165:322, 1985). In practice, a preferred medium must be foundexperimentally for each genotype. Typically in such an experimentalprocedure a selection of basal media, sucrose concentrations, and growthregulator types and concentrations are combined in a factorialarrangement. Immature embryos from each genotype to be tested are placedonto medium representing each factorial combination. Initiationfrequencies are scored for each medium and the ones producing thehighest scores are used in a second round of experimentation. In thissecond round, the selected media combinations are further optimized forthe individual genotypes by fine-tuning the growth-regulator type andconcentration, and sucrose concentration. For example, Table I belowindicates the preferred medium for the initiation of embryogenic callusfor several of the genotypes disclosed in this invention. TABLE IGenotype Initiation Medium LH51 MS basal medium, G10 amendments, 6%sucrose, 5 mg/L dicamba CG00716 JMS basal medium, G5 amendments, 9%sucrose, 5 mg/L dicamba CG00526 D basal medium, G8 amendments, 2%sucrose, 5 mg/L chloramben CG00642 JMS basal medium, G5 amendments, 10%sucrose, 4 mg/L dicamba LH82 LM basal medium, G6 amendments, 4% sucrose,0.2 mg/L 2,4-D CG00689 MS basal medium, G1 amendments, 6% sucrose, 0.5mg/L 2,4-D CGNC4206 MS basal medium, G1 amendments, 6% sucrose, 0.52,4-D CG00629 D basal medium, G8 amendments, 2% sucrose, 5 mg/Lchloramben (H99xFR16)xPa91 JMS basal medium, G5 amendments, 2% sucrose,1 mg/l dicamba Hi II JMS basal medium, G5 amendments, 2% sucrose, 10mg/L silver nitrate, 5 mg/L dicamba

The basal media formulas used for the initiation of embryogenic callusof the genotypes in Table I may be found in the following. citations andis herein incorporated by reference: “D”, (Duncan et al., Planta165:322, 1985); “KM”, (Kao et al., Planta 126:105, 1975); “LM”, (Litvayet al., Plant Cell Reports 4:325, 1985); “MS”, (Murashige et al,Physiologia Plantarum 15:473, 1962). The basal medium “JMS” is themedium known as “SH” (Schenk et al., Can. J. Bot. 50:199, 1972) modifiedby replacing the inorganic nitrogen compounds with those found in “MS”(Murashige et al., supra, 1962)

The formulas of the amendments used for the initiation of embryogeniccallus of the genotypes in Table II are found in the following table.The amendments used with the “KM” basal medium were as described in Kaoet al., supra, 1975). TABLE II Amendment Formulas, per Liter of MediumG1 G5 G6 G8 G10 Component mg Nicotinic Acid 0.5 5.0 0.5 0.2 0.5Pyridoxine-HCl 0.5 0.5 0.1 0.2 0.5 Thiamine-HCl 0.1 5.0 0.1 0.5 0.1Glycine 2.0 — — — 2.0 myo-Inositol 100 100 100 — 100 Choline HCl — — —0.1 — Riboflavin — — — 0.1 — Biotin — — — 0.1 — Folic Acid — — — 0.5 -CaPantothenate — — — 0.1 — Cyanocobalamin — — — 0.014 — Caseinhydrolysate 100 100 100 — — Proline 2800 2800 2800 — —

Often, the preferred “maintenance” medium must be determinedexperimentally, as is done for “initiation”. Table III describes the“maintenance” medium found to be useful for several of the genotypesdisclosed in the present invention. TABLE III Genotype MaintenanceMedium LH51 MS basal medium, G10 amendments, 3% sucrose, 0.25 mg/L 2,4-DCG00716 N6 basal medium, 25 mM proline, 100 mg casein hydrolysate, 2%sucrose, 1.5 mg/L 2,4-D CG00526 D basal medium, G8 amendments, 2%sucrose, 0.5 mg/L 2,4-D LH82 LM basal medium, G6 amendments, 3% sucrose,3 mg/L chloramben CG00689 MS basal medium, G10 amendments, 2% sucrose,1.5 mg/L 2,4-D CGNC4206 D basal medium, G8 amendments, 2% sucrose, 1.22,4-D (H99xFR16)xPa91 D basal medium, G4 amendments, 2% sucrose, 0.5mg/l 2,4-D Hi II N6 basal medium, G5 amendments, 2% sucrose, 1 mg/L2,4-DIn Table III, “N6” basal medium refers to that described in Chu et al.Scientia Sinica, XVIII: 659, 1975. The formulas for the amendments usedin “maintenance” medium may be found in Table II.

According to the present invention, nucleic acid sequences or genes ofinterest are delivered to the immature embryos within the “initiation”step of the development of an embryogenic callus, i.e., within about 14days of the placement of the immature embryos on a nutrient mediumcapable of supporting the intiation and development of embryogeniccallus. After gene delivery and initiation of embryogenic callus, theembryogenic callus is transferred to a “maintenance” medium forsubculture in either the presence or absence of a selection agent. Inanother embodiment of the invention, Type I callus developed using themethods above may also be used as a recipient of the nucleic acidsequences or genes of interest. In this instance, after delivery of thenucleic acid of interest, the Type I embryogenic callus is normallytransferred to fresh “maintenance” medium, with or without a selectionagent.

There are available many types of microprojectile bombardment devices,all working on essentially the same principle of accelerating micrometersize particles sufficient to cause penetration into the target tissuesand cells. Since it is known that bombardment devices based on gunpowderdo not work for the present invention, the preferred devices are thosethat use some form of gas expansion for the accelerating force for themicroprojectiles such as air, carbon dioxide, nitrogen, water vapor orhelium. The most preferred device for use in the claimed method is onebased on compressed helium such as the DuPont PDS-1000/He.

For the bombardment of Type I embryogenic callus, the callus must besubdivided into smaller pieces. This can be achieved by chopping,maceration, dissection or other mechanical means. It is also possible tosubdivide the callus through enzymatic means. Enzymes that digest thecell wall or cell-wall components may be used to reduce the integrity ofthe callus mass. Such enzymes include cellulases, macerases, pectinases,hemi-cellulases and others well known in the art. After the callus issubdivided, it is rinsed several times with liquid culture medium.

In preparation for gene delivery by microprojectile bombardment, eitherthe immature embryos or Type I embryogenic callus may optionally bepre-treated with an osmotically-active agent to plasmolyze the cells fora period from about 1 to about 24 hours, preferably from about 1 toabout 12 hours, most preferably from about 1 to about 6 hours.Typically, the recipient material is treated with a concentration ofsucrose that produces an osmotic pressure in the medium that is greaterthan that in the recipient material. The concentration of sucrose mayrange from about 2 to about 18%, preferably from about 6 to about 12%.It is also within the scope of this invention that otherosmotically-active agents may be used in concentrations sufficient tocause the plasmolysis of the cells of the recipient material, such assorbitol, glucose, mannitol and various molecular weight ranges ofpolyethylene glycol. The recipient material is kept in the presence ofthe osmotically-active agent after gene delivery for a period of about 1to about 24 hours, preferably from about 3 to about 18 hours, mostpreferably from about 10 to about 18 hours.

In a preferred embodiment of the invention, the set-up and use of themicroprojectile bombardment device, as well as the targeting of therecipient material, is described below. Other arrangements are alsopossible.

The DNA is prepared for microprojectile bombardment by chemicalprecipitation in the presence of micrometer size gold, essentiallyaccording to the published procedure of DuPont. In addition to gold,other dense particles of micrometer size may be used, such as tungstenor platinum. In one modification of the DuPont procedure, the particlesthemselves are first prepared by suspending them in water andsonicating. The sonicated particles are then pelleted by centrifugationand resuspended in an aqueous solution of 50% glycerol. Particlesprepared in this way are then aliquoted into individual tubes containingapproximately 3 mg of gold particles per tube in a volume of 50 ul. DNAis added to each tube in varying amounts, depending upon the number ofplasmids to be used, their sizes and the final concentration of DNAdesired. For example, in a typical preparation to include four plasmidsfor use in the present invention, 2 ug of pCIB3089, 2 ug pCIB4436, 3 ugpCIB4433 and 4 ug pCIB4430 are added to each tube of aliquoted goldparticles. Next, about 50 ul of 2.5 M CaCl2 and about 20 ul of 1Mspermidine are added, in that order, to each tube while vortexing forabout 3 minutes. The DNA/gold complex is then gently centrifuged. Thesupernatant is removed, the particles are washed once with 250 ul ofabsolute ethanol, pelleted again and then resuspended in about 75 ul bffresh absolute ethanol. Each tube prepared in this way is enough of theDNA/gold complex for six “shots” with the PDS-1000/He. Ten ul of thewell-suspended DNA/gold complex is pipetted onto the macrocarrier sheetin a vibration-free environment.

In the PDS-1000/He device, a burst of helium is released by rupture of aplastic disk which is available in different pressure grades. Forexample, single disks, or combinations of disks, can be obtained whichrupture at 200, 450, 650, 900, 1100 1350, 1550, 1800, 2000 and 2200pounds per square inch of helium. This burst of gas propels themacrocarrier sheet, which is stopped by a stainless steel screen. Thescreen may be of different mesh sizes, such as 10×10, 16×16, 24×24, etc.Other settings are the macrocarrier flight distances, gap distance, andparticle flight distance. These settings are described in detail in themanufacturer's user's manual. Typically, a gap distance of about 5.5 mm,a macrocarrier flight distance of about 10 mm and a particle flightdistance of about 6 to 9 cm is-used. In addition, a screen or baffle maybe inserted within the particle flight distance between the stoppingscreen and the target plate. Such a screen or baffle disturbs the shockwave from the expanding gas thereby reducing damage to the target. Inone example, stainless steel screens with an opening of about 100 um isused. Other opening sizes and material composition may be used.

The immature embryos or Type I embryogenic callus may be arranged on thetarget plate in different patterns. Through a series of experiments,optimized patterns were developed for immature embryos. In one optimizedpattern, the immature embryos are arranged in a circular pattern, thecircle being about 2 cm in diameter. The immature embryos are placed onthe periphery of the circle. Approximately 36 immature embryos areplaced onto each target plate. Furthermore, the target plate may beangled relative to the microcarrier launch assembly. This ensuresmaximum saturation of the basipetal portion of the immature embryo bythe particle spread. It is the basipetal portion of the immature embryothat gives rise to the embryogenic response.

In one example of the bombardment of Type I embryogenic callus, thecallus is placed on the periphery of a circle about 1 cm diameter on anutrient medium. The mechanical settings of the bombardment device maybe placed at positions similar or identical to the settings recitedabove for the bombardment of immature embryos.

It should be noted that the target pattern and gun settings areinterrelated. In other words, the use. of other mechanical settings onthe microprojectile bombardment device can produce other optimalarrangements of the recipient tissue on the target plate. Othercombinations of mechanical settings and target patterns are within thescope of the invention.

The recombinant DNA molecules of the invention also can include a markergene to facilitate selection in recombinant plant cells.

Examples of markers include resistance to a biocide such as anantibiotic, e.g. kanamycin, hygromycin, chloramphenicol, paramomycin,methotrexate and bleomycin, or a herbicide such as imidazolones,sulfonylureas, glyphosate, phosphinothricin (PPT), glufosinate, orbialaphos. Marker genes are well known in the art. In one embodiment ofthe invention the selection agent phosphinothricin is used inconjunction with the selectable marker gene known as bar, which encodesfor the enzyme phosphinothricin acetyltransferase. This enzymeacetylates the phosphinothricin molecule, thereby rendering it non-toxicto plant cells. The bar gene may be operably linked to a constitutivepromoter such as the CaMV 35S promoter; the CaMV 19S promoter; A.tumefaciens promoters such as octopine synthase promoters, mannopinesynthase promoters, nopaline synthase promoters, or other opine synthasepromoters; ubiquitin promoters, actin promoters, histone promoters andtubulin promoters. Other promoters may also be used. In one embodimentof the present invention, the bar coding sequence is operably linked tothe CaMV 35S promoter.

Selection of transformed cells in vitro is accomplished by including theselection agent of interest in the medium used to induce and support theestablishment of embryogenic callus. Only cells in which the selectablemarker is integrated into the chromosome and is expressed, i.e., aretransformed, will survive the selection agent. Over time, the cells willgrow into an embryogenic callus in the presence of the selection agenteventually reaching a mass sufficient for regeneration of whole, fertileplants. Typically, such embryogenic callus can be maintained for longperiods of.time in the presence of selection agent and still retain itsability to produce whole, fertile plants. The minimum time required toobtain an embryogenic callus of sufficient mass under selection pressurecan range from about 2 weeks to about 24 weeks, more preferably about 4weeks to about 20 weeks, most preferably about 8 weeks to about 12weeks.

Transformed cells may also be selected in vitro through visual means. Inorder to accomplish this, a scorable marker is generally used. Examplesof scorable markers would be the regulatory or structural genescontrolling anthocyanin biosynthesis, GUS (beta-glucuronidase),luciferase, opine synthetases, thaumatin, beta-galactosidase, uniquesynthetic epitopes designed for easy detection by ELISA,phycobiliproteins and various fluorigenic substances. In a particularembodiment of the present invention the use is made of coding sequencesfor the anthocyanin regulatory genes known in the art as C1 and B-Peru(Goff et al., EMBO Journal, 9: 2517, 1990). Such coding sequences,operably linked to one or more of the several constitutive promoterslisted above, can be used to isolate transformants on the basis of thered pigmentation of cells transformed with such-genes.

Fertile transformed plants may be regenerated from isolated transformedembryogenic callus by several means. In general, the transformedembryogenic callus is transferred to a nutrient medium devoid of anauxin-type phytohormone, or is passaged through a series of nutrientmedia with diminishing concentrations of phytohormone. Otherphytohormones may be used during the regeneration step, such ascytokinins (both natural and synthetic) and gibbetellins. In someinstances, inhibitors of phytohormone action may also be used, such assilver nitrate, ancymidol or TIBA. Other amendments to the nutrientmedium for regeneration such as activated charcoal and various gellingagents are also known in the art.

In one example of the present invention, embryogenic callus was removedfrom maintenance medium containing PPT after 12 weeks and placed onregeneration medium containing MS basal medium, 3% sucrose, 0.25 mg/L2,4-D and 5 mg/L benzyladenine. After 2 weeks the embryogenic callusbegan to regenerate and was transferred to MS basal medium with 3%sucrose. Plantlets are obtained during incubation under light. Suchplants may be transferred to a greenhouse environment after sufficientroot mass has developed.

In another embodiment of the invention, transformed embryogenic callusis transferred from maintenance medium containing phosphinothricin toregeneration medium consisting of MS medium, 3%. sucrose and alsocontaining phosphinothricin. Regeneration under such selectiveconditions also produces plantlets during incubation under light, whichagain may be transferred to a greenhouse environment after sufficientroot mass has developed.

As will be evident to one of skill in the art, now that a method hasbeen provided for the stable transformation of maize according to theclaimed method, any gene of interest can be used in the methods of theinvention. For example, a maize plant can be engineered to expressdisease and insect resistance genes, genes conferring nutritional value,genes to confer male and/or female sterility, antifungal, antibacterialor antiviral- genes, and the like. Likewise, the method can be used totransfer any nucleic acid to control gene expression. For example, theDNA to be transferred could encode antisense RNA.

The present invention also encompasses the production by the disclosedmethod of transformed maize plants and progeny containing a gene orgenes which encode for and express insecticidal proteins. Such genes maybe derived from the genus Bacillus, for example Bacillus thuringiensis.In a particular embodiment of the present invention is the use of theclaimed method to produce transformed maize plants containing a gene orgenes whose nucleic acid sequence has been altered so as to be optimizedfor expression in maize. A complete description of the creation of saidgene or genes may be found in U.S. Ser. No. 951,715 which is hereinincorporated by reference. A summary of that disclosure- is given below.

A nucleic acid sequence of interest in the present invention includesone which encodes the production of an insecticidal toxin, preferably apolypeptide sharing substantially the. amino acid sequence of aninsecticidal crystal protein toxin normally produced by Bacillusthuringiensis (BT). The synthetic gene may encode a truncated orfull-length insecticidal protein (IP) Especially preferred are syntheticnucleic sequences which encode a polypeptide effective against insectsof the order Lepidoptera and Coleoptera, and synthetic nucleic acidsequences which encode a polypeptide having an amino acid sequenceessentially the same as one of the crystal protein toxins of Bacillusthuringiensis variety kurstaki, HD-1.

The present invention provides the use of synthetic nucleic acidsequences to yield high level expression of active insecticidal proteinsin maize plants. The synthetic,nucleic acid sequences of the presentinvention have been modified to resemble a maize gene in terms of codonusage and G+C content. As a result of these modifications, the syntheticnucleic acid sequences of the present invention do not contain thepotential processing sites which are present in the native gene. Theresulting synthetic nucleic acid sequences (synthetic BT IP codingsequences) and plant transformation vectors containing this syntheticnucleic acid sequence (synthetic BT IP genes) result in surprisinglyincreased expression of the synthetic BT IP gene, compared to the nativeBT IP gene, in terms of insecticidal protein production in plants,particularly maize. The high level of expression results in maize cellsand plants that exhibit resistance to lepidopteran insects, preferablyEuropean Corn Borer and Diatrea saccharalis, the Sugarcane Borer.

For example, the maize codon usage table described in Murray et al.,Nucleic Acids Research, 17:477 1989, the disclosure of which isincorporated herein by reference, was used to reverse translate theamino acid sequence of the toxin produced by the Bacillus thuringiensissubsp. kurstaki HD-1 cryIA(b) gene, using only the most preferred maizecodons. This sequence was subsequently modified to eliminate unwantedrestriction endonuclease sites, and to create desired restrictionendonuclease sites. These modifications were designed to facilitatecloning of the gene without appreciably altering the codon usage or themaize optimized sequence. During the cloning procedure, in order tofacilitate cloning of the gene, other modifications were made in aregion that appears especially susceptible to errors induced duringcloning by the polymerase chain reaction (PCR).

In a preferred embodiment of the present invention, the protein producedby the synthetic nucleic acid sequence of interest is effective againstinsects of the order Lepidoptera or Coleoptera. In a more preferredembodiment, the polypeptide encoded by the synthetic nucleic acidsequence of interest consists essentially of the full-length or atruncated amino acid sequence of an insecticidal protein normallyproduced by Bacillus thuringiensis var. kurstaki HD-1. In a particularembodiment, the synthetic DNA sequence encodes a polypeptide consistingessentially of a truncated amino acid sequence of the BT CryIA(b)protein.

The present invention also encompasses the use of maize optimized codingsequences encoding other polypeptides, including those of other Bacillusthuringiensis insecticidal polypeptides or insecticidal proteins fromother sources. For example, cryIB genes from Bacillus thuringiensis canbe maize optimized, and then stably introduced into maize plants. It isalso within the scope of this invention that the nucleic acid sequencesof interest which encode insecticidal proteins may be either in thenative or synthetic forms, optimized for expression in maize, andderived from any species of the genus Bacillus.

The insecticidal proteins produced by the nucleic acid sequences ofinterest in the present invention are expressed in a maize plant in anamount sufficient to control insect pests, i.e. insect controllingamounts. It is recognized that the amount of expression of insecticidalprotein in a plant necessary to control insects may vary depending uponspecies of plant, type of insect, environmental factors and the like.Generally, the insect population will be kept below the economicthreshold which varies from plant to plant. For example, to controlEuropean corn borer in maize, the economic threshold is 0.5eggmass/plant which translates to about 10 larvae/plant.

In the present invention, the coding sequence of the nucleic acids ofinterest is a synthetic maize-optimized gene under the control ofregulatory elements such as promoters which direct expression of thecoding sequence. Such regulatory elements, for example, include monocotor maize and other monocot functional promoters to provide expression ofthe gene in various parts of the maize plant.

The regulatory element may be constitutive. That is, it may promotecontinuous and stable expression of the gene. Such promoters include butare not limited to the CaMV 35S promoter; the CaMV 19S promoter; A.tumefaciens promoters such as octopine synthase promoters, mannopinesynthase promoters, nopaline synthase promoters, or other opine synthasepromoters; ubiquitin promoters, actin promoters, histone promoters andtubulin promoters.

The regulatory element may also be a tissue-preferential ortissue-specific promoter. The term “tissue-preferred promoter” is usedto indicate that a given regulatory DNA sequence will promote a higherlevel of transcription of an associated structural gene or DNA codingsequence, or of expression of the product of the associated gene asindicated by any conventional RNA or protein assay, or that a given DNAsequence will demonstrate some differential effect; i.e., that thetranscription of the associated DNA sequences or the expression of agene product is greater in some tissue than in all other tissues of theplant. Preferably, the tissue-preferential promoter may direct higherexpression of the synthetic gene in leaves, stems, roots and/or pollenthan in seed. “Tissue-specific promoter” is used to indicate that agiven regulatory DNA sequence will promote transcription of anassociated coding DNA sequence essentially entirely in one or moretissues of a plant, or in one type of tissue, e.g. green tissue, whileessentially no transcription of that associated coding DNA sequence willoccur in all other tissues or types of tissues of the plant. Numerouspromoters whose expression are known to vary in a tissue specific mannerare known in the art. One such example is the maize phosphoenol pyruvatecarboxylase (PEPC), which is green tissue-specific. See, for example,Hudspeth, R.L. and Grula, J. W., Plant Molecular Biology 12:579-589,1989. Other green tissue-specific promoters include chlorophyll a/bbinding protein promoters and RubisCO small subunit promoters.

The regulatory element may also be inducible, such as by heat stress,water stress, insect feeding or chemical induction, or may bedevelopmentally regulated.

In one preferred nucleic acid of interest, the regulatory element is apith-preferred promoter isolated from a maize TrpA gene.

That is, the promoter.in its native state is operatively associated witha maize tryptophan synthase-alpha subunit gene (hereinafter “TrpA”). Theencoded protein has a molecular mass of about 38kD. Together withanother alpha subnit and two beta subunits, TrpA forms a multimericenzyme, tryptophan synthase.

Each subunit can operate separately, but they function more efficientlytogether.

The nucleic acids of interest in the present invention also includepurified pollen-specific promoters obtainable from a plantcalcium-dependent phosphate kinase (CDPK) gene. That is, in its nativestate, the promoter is operably linked to a plant CDPK gene. In apreferred embodiment, the promoter is isolated from a maize CDPK gene.By “pollen-specific,” it is meant that the expression of an operativelyassociated structural gene of interest is substantially exclusively(i.e. essentially entirely) in the pollen of a plant, and is negligiblein all other plant parts. By “CDPK,” it is meant a plant protein kinasewhich has a high affinity for calcium, but not calmodulin, and requirescalcium, but not calmodulin, for its catalytic activity.

In another nucleic acid of interest, the regulatory element is aroot-preferential promoter. A complete description of such a rootpromoter and the methods for finding one may be found in U.S. Ser. No.508,207 filed Apr. 12, 1990, the relevant parts of which are hereinincorporated by reference. Briefly, a root-preferential promoter wasisolated from a gene whose cDNA was found by differential screening of aCDNA library from maize. A CDNA clone so obtained was used to isolate ahomologous genomic clone from maize. The protein encoded by the isolatedclone was identified as a metallothionein-like protein.

Maize is easily hybridized because of the physical distance between thetassel (male part) and the ear (female part). The method ofhybridization first involves the development of inbred lines. Inbredlines are maize plants that are essentially the same genetically fromgeneration to generation. Inbreds are produced by taking the pollen fromone maize plant and transferring the pollen to the silk of a receptivemaize ear of that same plant. Selections for uniformity and agronomicperformance are made and the process is repeated until the.seeds fromthe ears of the plants produce genetically the same plants and. the lineis pure. A hybrid maize plant is produced by crossing one elite inbredmaize plant with one or more other, genetically different and diverse,inbred maize plant. The crossing consists of taking the pollen from oneinbred elite maize plant and transferring the pollen to the silk of areceptive ear of the other elite inbred maize plant. The seed fromcrossing of two inbreds is a first generation hybrid and is called a F1.The F1 of commercially valuable inbreds have better yields,standability, and improvement in other important characteristics thaneither of the parents. This phenomenon is called hybrid vigor.

In the present invention, commercially-valuable inbred lines of maizeare directly transformed through the disclosed methods of deliveringnucleic acid sequences of interest to either immature zygotic embryosobtained from such lines or Type I embryogenic callus derived fromimmature zygotic embryos of such lines. The ability to directlytransform maize lines of commercial value is a distinct advantage of theclaimed invention in that the generations of backcrossing required whenthe starting material is not commercially valuable can be avoided,thereby reducing the time and cost of commercialization. Alternatively,the present invention also discloses the direct transformation ofhybrids of inbred lines using the claimed methods.

Many hybrid crosses have been successfully made using the transformed,commercially-valuable plants of the claimed invention. For example, thetransformed genotype CG00526 of Example 2, below, has been crossed togenotypes CG00689, CG00716, CG00661, CG00642, and LH82 thereby creatinghybrids possessing insecticidal activity.

Using the methods of the present invention any hybrid expressing a geneof interest can be created by transforming an inbred line with the geneof interest and using such transformed line to create the hybrid. Atransformed hybrid may also be obtained according to the presentinvention by directly transforming either immature zygotic embryosobtained from said hybrid plant or by transforming Type I embryogeniccallus derived from immature zygotic embryos obtained from said hybridplant.

In another embodiment of the claimed invention, it is also possible toproduce maize plants that have an altered phenotype of anthocyaninpigmentation. This can be accomplished through the use of the disclosedchimeric genes for the constitutive promotion of the genes known as C1and B-Peru. That activation of the biosynthetic pathway for anthocyanincan be achieved in this way in embryogenic callus was reported by Goffet al., EMBO Journal, 9: 2517-2522, 1990. In the present invention, theabove said genes were used to produce plants and progeny according tothe claimed method whose color phenotype was altered.

Commercially-valuable maize genotypes having altered-color phenotype mayhave benefit to the process of plant breeding. For example, the use ofthe monoploid inducing gene known as ig (Kermicle, Science166:1422-1424, 1969) can be used to create a haploid having a paternalnuclear constitution. Monoploid inducers creating a haploid having amaternal nuclear constitution ate also known (for example, Coe, TheAmerican Naturalist, XCIII: 381-382, 1959). Because of the low frequencyof such an event, it would be advantageous to have an easily screenedcolor phenotype which would allow the identification of the haploids. Byusing the claimed method and genes of interest, it is possible to obtaina transformed maize line having pigmented seeds, which can be used witha monoploid inducing line. Haploid seed can then be identified by eitherthe presence or absence of seed pigmentation, depending upon thegenotypes and crossing methods used.

Since a variety of altered color phenotypes can be produced by thepresent invention, examples of which are described below, it is furtherenvisioned that other uses in plant breeding may be found for theclaimed plants. As another example of such utility, it is possible tolink operably, molecularly, biochemically or genetically, or anycombination thereof, the expression of the altered color phenotype withthe expression of the insecticidal activity produced by transformationof maize according to the claimed methods. Such a link would allowrapid, visual identification of plants within a segregating populationof plants and possesing the gene or gene products. Linkages of thealtered color phenotype and genotype to other traits of agronomicinterest are also envisioned. The ability to perform such identificationwould translate intq reduced costs and time for the plant breeder.

EXAMPLES

The following examples further describe the materials and methods usedin carrying out the invention and the subsequent results.

They are offered by way of illustration, and their recitation should notbe considered as a limitation of the claimed invention.

Example 1

Bioassay of Transformed Maize for Insecticidal Activity and Quantitationof an Insecticidal Protein.

Transformed plants were assayed for insecticidal activity and thepresence of a BT protein resulting from the expression of themaize-optimized coding sequence of a synthetic BT gene. The procedure issimilar for any maize plant transformed with a BT gene but is describedhere using as an example a cryIA(b) gene, its expressed product, andresistance to European corn borer.

Insecticidal activity was determined by insect bioassay. One to four 4cm sections are cut from an extended leaf of a transformed maize plant.Each leaf piece is placed on a moistened filter disc in a 50×9 mm petridish. Five neonate European corn borer larvae are placed on each leafpiece. Since each plant is sampled multiple times this makes a total of5-20 larvae per plant. The petri dishes are incubated at 29.5° C. andleaf feeding damage and mortality data are scored at 24, 48, and 72hours.

Quantitative determination of a cryIA(b) IP in the leaves of transgenicplants is performed using enzyme-linked immunosorbant assays (ELISA) asdisclosed in Clark M F, Lister R M, Bar-Joseph M: ELISA Techniques. In:Weissbach A, Weissbach H (eds) Methods in Enzymology 118:742-766,Academic Press, Florida (1986).

Immunoaffinity purified polyclonal rabbit and goat antibodies specificfor the B. thuringiensis subsp. kurstaki IP were used to determine ng IPper mg soluble protein from crude extracts of leaf samples. Thesensitivity of the double sandwich ELISA is 1-5 ng IP per mg solubleprotein using 50 ug of total protein per ELISA microtiter dish well.Corn extracts were made by grinding leaf tissue in gauze lined plasticbags using a hand held ball-bearing homogenizer (AGDIA, Elkart IN.) inthe presence of extraction buffer (50 mM Na2CO3 pH 9.5, 100 mM NaCl,0.05% Triton, 0.05% Tween, 1 mM PMSF and 1 pM leupeptin). Proteindetermination was performed using the Bio-Rad (Richmond, CA) proteinassay.

Example 2

Transformation of the CG00526 Genotype of Maize by Direct Bombarding ofImmature Zygotic Embryos and Isolation of Transformed Callus with theUse of Phosphinothricin as a selection agent.

Immature embryos for experiment KC-65 were obtained approximately 14days after self-pollination. The immature zygotic embryos were dividedamong different target plates containing medium capable of inducing andsupporting embryogenic callus formation at 36 immature embryos perplate. The immature zygotic embryos were bombarded with a mixture of theplasmids pCIB3064 and pCIB4431 using the PDS-1000/He device from DuPont.The plasmids were precipitated onto 1 um gold particles essentiallyaccording to the published procedure from DuPont, as described.above.Each target plate was shot one-time with the plasmid and goldpreparation. Since the plasmid pCIB3064 contained a chimeric gene codingfor resistance to phosphinothricin this substance was used to selecttransformed cells in vitro. This selection was applied at 3 mg/L one dayafter bombardment and maintained for a total of 12 weeks. Theembryogenic callus so obtained was regenerated in the absence of theselection agent phosphinothricin. Plants were obtained from one isolatedline of embryogenic callus and given the Event Number 176. All plantswere tested by the chlorophenol red (CR) test for resistance to PPT asdescribed in U.S. patent application Ser. No. 759,243, filed Sep. 13,1991, the relevant portions of which are hereby incorporated herein byreference. This assay utilizes a pH sensitive indicator dye to showwhich cells are growing in the presence of PPT. Cells which grow producea pH change in the media and turn the indicator yellow (from red).Plants expressing the resistance gene to PPT are easily seen in thistest. Of the 38 plants regenerated for Event Number 176, eight werepositive in this test and 30 were negative. Plants positive by the CRtest were assayed by PCR for the presence of the synthetic BT gene. Ofthe eight positive plants from the CR test, 5 were positive for thepresence of the synthetic BT gene, 2 were negative and 1 died duringpropagation. These five remaining plants were bioassayed and found to beresistant to European Corn Borer. DNA was isolated from plant #11 usingstandard techniques and analysed by Southern blot analysis. It was foundto contain sequences which hybridize with probes generated from thesynthetic cryIA(b) gene and with a probe generated from the PAT gene.These results showed integration of these genes into the genome ofmaize. Plant 11 was shown by ELISA to contain 2,195 ng BT protein per mgsoluble protein in the leaf tissue, consistent with the use of theleaf-specific promoter from PEPC operably linked to a synthetic BT gene.Plants resistant to European Corn Borer and expressing the introduced BTgene are transformed.

Example 3

Transformation of the Hi II genotype of Maize by Direct Bombarding ofImmature Zygotic Embryos and Isolation of Transformed Callus without theUse of a Selection Agent.

Ear number ED42 was self-pollinated and immature zygotic embryos wereobtained approximately 10 days later. Two hundred and eighty eightimmature zygotic embryos were divided among 7 different target platescontaining a medium capable of inducing and supporting the formation ofembryogenic callus. After two days, the immature zygotic embryos weretransferred to the same medium but containing 12% sucrose. After 5hours, the immature zygotic embryos were bombarded with a mixture of theplasmids pCIB3089, pCIB4430, pCIB4433, pCIB4436 using the PDS-1000/Hedevice from DuPont. The plasmids were precipitated onto 1 um goldparticles essentially according to the published procedure from DuPont,as described above. The particles were delivered using burst pressuresof 450, 650 and 900 psi of helium. Each target plate was shot twice withthe plasmid and gold particle preparation. After overnight incubation,the immature embryos were transferred to fresh maintenance mediumcontaining 2% sucrose. Since the plasmids pCIB3089 and pCIB4436 containthe C1 and B-Peru genes which regulate anthocyanin production, theappearance of red, multicellular sectors on the developing embryogeniccallus was used to select and isolate transformed cells, eventuallyobtaining a homogeneous callus line. Embryogenic callus was regeneratedin both the absence and presence of the selection agentphosphinothricin, resistance to which was conferred by a chimeric genepresent in plasmid pCIB4433. Plants were obtained from a total of nineisolated embryogenic callus lines and were given the Event Numbers 197,198, 208, 211, 219, 255, 261, 281 and 284. Leaf tissue from plants fromeach event were assayed for resistance to European Corn Borer. Plantsfrom Event Numbers 208 and 211 were susceptible to European Corn Borerwhereas plants from Event Numbers 197, 198, 219, 255, and 261 wereresistant. All the plants that were resistant to European Corn Boreralso expressed the introduced, leaf-specific PEPC-promoted synthetic BTgene as evidenced by thd detection of BT protein using an ELISA assay.

Plants resistant to European Corn Borer and expressing the introduced BTgene are transformed.

Example 4

Transformation of the Hi II Genotype of Maize by Direct Bombarding ofImmature Zygotic Embryos and Isolation of Transformed Callus with theUse of Phosphinothricin as a Selection Agent.

Ear number ED47 was self-pollinated and immature zygotic embryos wereobtained approximately 10 days later. Approximately two hundred andsixty immature zygotic embryos were divided among 8 different targetplates containing a medium capable of inducing and supporting theformation of embryogenic callus. After two days, the immature zygoticembryos were transferred to the same medium but containing 12% sucrose.After 5 hours, the immature zygotic embryos were bombarded with amixture of the plasmids pCIB3089, pCIB4430, pCIB4433, pCIB4436 using thePDS-1000/He device from DuPont. The plasmids were precipitated onto 1 umgold particles essentially according to the published procedure fromDuPont, as described above. The particles were delivered using a burstpressure of 900 psi of helium. Each target plate was shot twice with theplasmid and gold particle preparation. After overnight incubation, -theimmature embryos were transferred to fresh maintenance medium containing2% sucrose. Since the plasmid pCIB4433 contained a chimeric gene codingfor resistance to phosphinothricin this substance was used to selecttransformed cells in vitro. The selection agent was applied at 10 mg/L14 days after gene delivery and increased to 20-40 mg/L afterapproximately one month. The embryogenic callus so obtained wasregenerated in the presence of the selection agent phosphinothricin.Plants were obtained from a total of eleven isolated embryogenic calluslines and were given the Event Numbers 220, 221, 222, 223, 225, 230,231, 232, 233, 269, 274. Plants from each event were assayed forresistance to European Corn Borer. Leaf tissue of plants from EventNumbers 220, 221, 222, 223, 225, 231 and 233 were resistant. All theplants that were resistant to European Corn Borer also expressed theintroduced, leaf-specific PEPC-promoted synthetic BT gene as evidencedby the detection of BT protein using an ELISA assay. Plants resistant toEuropean Corn Borer and expressing the introduced BT gene aretransformed. Plants from Event Numbers 230, 232, 269 and 274 were notcompletely tested.

Example 5

Transformation of the CG00526 Genotype of Maize by Bombarding of Type ICallus Derived from Immature Zygotic Embryos and Isolation ofTransformed Callus with the Use of Phosphinothricin as a SelectionAgent.

Type I callus was obtained from immature zygotic embryos using standardculture techniques. For gene delivery, approximately 300 mg of the TypeI callus was prepared by chopping with a scalpel blade, rinsing 3 timeswith standard culture media containing 18% sucrose and immediatelyplaced onto semi-solid culture medium again containing 18% sucrose.After approximately 4 hours, the tissue was bombarded using thePDS-1000/He Biolistic device from DuPont. The plasmids pCIB4430 andpCIB4433 were precipitated onto 1 um gold particles using the standardprotocol from DuPont. Approximately 16 hours after gene delivery thecallus was transferred to standard culture medium containing 2% sucroseand 10 mg/L phosphinothricin as Basta. The callus was subcultured onselection for 8 weeks, after which surviving and growing callus wastransferred to standard regeneration medium for the production ofplants.

Example 6

Transformation of the LH51 Genotype of Maize by Bombarding of Type ICallus Derived from Immature zygotic embryos and Isolation ofTransformed Callus with the Use of Phosphinothricin as a SelectionAgent.

Type I callus was obtained from immature zygotic embryos using standardculture techniques. For gene delivery, approximately 300 mg of the TypeI callus was prepared by chopping with a scalpel blade, rinsing 3 timeswith standard culture media containing 12% sucrose and immediatelyplaced onto semi-solid culture medium again containing 12% sucrose.After approximately 4 hours, the tissue was bombarded using thePDS-1000/He Biolistic device from DuPont. The plasmids pCIB4430 andpCIB4433 were precipitated onto 1 um gold particles using essentiallythe standard protocol from DuPont as described above. Approximately 16hours after gene delivery the callus was transferred to standard culturemedium containing 2% sucrose and 1 mg/L phosphinothricin as Basta. Thecallus was subcultured on selection for 8 weeks, after which survivingand growing callus was transferred to standard regeneration medium forthe production of plants.

Example 7

Transformation of the CG00526 Genotype of Maize by Direct Bombarding ofImmature Zygotic Embryos and Isolation of Transformed Callus with theUse of Phosphinothricin as a Selection Agent.

Ear numbers JS21, JS22, JS23, JS24 and JS25 were self-pollinated andimmature zygotic embryos were obtained approximately 10 days later.Approximately eight hundred and forty immature zygotic embryos weredivided among 14 different target plates containing a medium capable ofinducing and supporting the formation of embryogenic callus. Theimmature zygotic embryos were transferred immediately to the same mediumbut containing 12% sucrose. After 5 hours, the immature zygotic embryoswere bombarded with a mixture of the plasmids pCIB3089, pCIB4433,pCIB4436 using the PDS-1000/He device from DuPont. The plasmids wereprecipitated onto 1 um gold particles essentially according to thepublished procedure from DuPont, as described above. The particles weredelivered using a burst pressure of 1550 psi of helium. Each targetplate was shot twice with the plasmid and gold particle preparation.Since the plasmid pCIB4433 contained a chimeric gene coding forresistance to phosphinothricin this substance was used to selecttransformed cells in vitro. The selection agent was applied at 10 mg/Lon the day of gene delivery and increased to 40 mg/L after approximatelyone month. The embryogenic callus so obtained was regenerated in thepresence of the selection agent phosphinothricin. Plants were obtainedfrom a total of eight isolated embryogenic callus lines and were giventhe Event Numbers 187, 188, 191, 192, 193, 196, 228 and 229. Plants fromeach event were assayed for resistance to European Corn Borer. Plantsfrom Event Numbers 191 and 193 exhibited insect resistance in the pithin accordance with the use of the pith-preferred synthetic BT construct.All the plants that were resistant to European Corn Borer also expressedthe introduced chimeric BT gene as evidenced by the detection of BTprotein in the pith using an ELISA assay. Plants resistant to EuropeanCorn Borer and expressing the introduced BT gene are transformed.

Example 8

Transformation of the (H99xFR16)xPa9l Genotype of Maize by DirectBombarding of Immature Zygotic Embryos and Isolation of TransformedCallus with the Use of Phosphinothricin as a Selection Agent.

Ear numbers GP5 and JS26 were self-pollinated and immature zygoticembryos were obtained approximately 10 days later. Approximately threehundred and thirty immature zygotic embryos were divided among 5different target-plates containing a medium capable of inducing andsupporting the formation of embryogenic callus. After two days theimmature zygotic embryos were transferred to the same medium butcontaining 12% sucrose. After approximately 5 hours, the immaturezygotic embryos were bombarded with a mixture of the plasmids pCIB3089,pCIB4430, pCIB4433, pCIB4436 using the PDS-1000/He device from DuPont.The plasmids were precipitated onto 1 um gold particles essentiallyaccording to the published procedure from DuPont, as described above.The particles were delivered using a burst pressure of 1300 psi ofhelium. Each target plate was shot twice with the plasmid and goldparticle preparation. Since the plasmid pCIB4433 contained a chimericgene coding for resistance to phosphinothricin this substance was usedto select transformed cells in vitro. The selection agent was applied at10 mg/L 3 weeks after the day of gene delivery. The embryogenic callusso obtained was regenerated. Plants were obtained from three isolatedembryogenic callus lines and were given the Event Numbers 242, 247 and260. Plants from each event were assayed for resistance to European CornBorer. Plants from Event Numbers 247 and 260 exhibited insect resistanceindicating that they were transformed.

Example 9

Transformation of the CGO0526 Genotype of Maize by Direct Bombarding ofImmature zygotic embryos and Isolation of Transformed Callus with theUse of Phosphinothricin as a Selection Agent.

Immature zygotic embryos for. the experiment KM-124 were obtainedapproximately 14 days after self-pollination. Approximately one hundredand five immature zygotic embryos were divided among 4 different targetplates containing a medium capable of inducing and supporting theformation of embryogenic callus. The immature zygotic embryos werebombarded with a mixture of the plasmids pCIB4421 and pCIB4433 using thePDS-1000/He device from DuPont.

The plasmids were-precipitated onto 1 um gold particles essentiallyaccording to the published procedure from DuPont, as described above.The particles were delivered using a burst pressure of 1550 psi ofhelium. Each target plate was shot once with the plasmid and goldparticle preparation. Since the plasmid pCIB4433 contained a chimericgene coding for resistance to phosphinothricin this substance was used,as Basta, to select transformed cells in vitro. The selection agent wasapplied at 5 mg/L one day after gene delivery and maintained for a totalof 12 weeks. The embryogenic callus so obtained was regenerated in theabsence of the selection agent phosphinothricin. Plants were obtainedfrom one isolated embryogenic callus line and was given the Event Number268. Plants were assayed for resistance to European Corn Borer. One ofthe 5 plants obtained is resistant to European Corn Borer and istransformed.

Example 10

Color Phenotypes Exhibited by Plants Transformed with the C1 and B-PeruCoding Sequences According to the Claimed Methods

Both genotypes Hi II and CG00526 were transformed with the C1 and B-Peruchimeric genes recited above in FIGS. 2 and 7. A variety of stablyexpressed altered color phenotypes were obtained, a partial listing ofwhich -appears in Table IV, below. TABLE IV BT Event Number ColorPhenotype 197 Red roots, red anthers 208 Red roots 211 Red roots 213 Redstripe shoot 204 Red anthers, pink silks 210 Red stripe shoot, red root,red anther, red silk, red embryo 239 Red shoot, red root, red silk,normal embryo 207 Red anthers, red silks

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1-82. (canceled)
 82. A fertile transgenic Zea mays plant comprising apreselected DNA sequence encoding a Bacillus thuringiensis endotoxin,wherein the preselected DNA sequence is adjusted to be more efficientlyexpressed in maize than the native B. thuringiensis DNA sequenceencoding said endotoxin, and wherein said preselected DNA is heritable.83. The transgenic Zea mays plant of claim 82 wherein the preselectedDNA sequence comprises an increased G+C content of the degenerate thirdbase of the codons.
 84. The transgenic Zea mays plant of claim 82wherein the preselected DNA sequence comprises a sequence encoding theHD73 endotoxin of Bacillus thuringiensis.
 85. A seed produced by thetransgenic Zea mays plant of claim 82, 83 or 84 which comprises saidpreselected DNA sequence.
 86. The transgenic Zea mays plant of claim 82wherein the preselected DNA sequence encodes a truncated Bacillusthuringiensis endotoxin.
 87. The transgenic Zea mays plant of claim 82wherein the preselected DNA sequence comprises a promoter.
 88. A fertileinbred transgenic Zea mays plant comprising a preselected DNA sequenceencoding a Bacillus thuringiensis endotoxin, wherein the preselected DNAsequence is adjusted to be more efficiently expressed in maize than thenative B. thuringiensis DNA sequence encoding said endotoxin and whereinthe preselected DNA sequence is heritable.
 89. A fertile hybridtransgenic Zea mays plant comprising a preselected DNA sequence encodinga Bacillus thuringiensis endotoxin, wherein the preselected DNA sequenceis adjusted to be more efficiently expressed in maize than the native B.thuringiensis DNA sequence encoding said endotoxin, and wherein thepreselected DNA sequence is heritable.
 90. A fertile transgenic Zea maysplant comprising a preselected heritable DNA sequence encoding aBacillus thuringiensis endotoxin, wherein the preselected DNA sequenceis adjusted to be more efficiently expressed in maize than the native B.thuringiensis DNA sequence encoding said endotoxin, and wherein thepreselected DNA sequence further comprises a selectable marker gene or areporter gene.
 91. The transgenic Zea mays plant of claim 90 wherein thepreselected DNA sequence comprises a sequence encoding the HD73endotoxin of Bacillus thuringiensis.
 92. The transgenic Zea mays plantof claim 90 wherein the preselected DNA sequence comprises a sequenceencoding the HD1 endotoxin of Bacillus thuringiensis.
 93. A seedproduced by the transgenic Zea mays plant of claim 90, 91 or 92 whichcomprises said preselected DNA sequence.
 94. The transgenic Zea maysplant of claim 90 wherein the DNA sequence encodes a truncated Bacillusthuringiensis endotoxin.
 95. The transgenic Zea mays plant of claim 90wherein the preselected DNA sequence further comprises a promoteroperably linked to said DNA sequence encoding said endotoxin and apromoter operably linked to said selectable marker gene.
 96. A fertileinbred transgenic Zea mays plant comprising a preselected heritable DNAsequence encoding a Bacillus thuringiensis endotoxin, wherein thepreselected DNA sequence is adjusted to be more efficiently expressed inmaize than the native B. thuringiensis DNA sequence encoding saidendotoxin, and wherein the preselected DNA sequence further comprises aselectable marker gene or a reporter gene.
 97. A fertile hybridtransgenic Zea mays plant comprising a preselected heritable DNAsequence encoding a Bacillus thuringiensis endotoxin, wherein thepreselected DNA sequence is adjusted to be more efficiently expressed inmaize than the native B. thuringiensis DNA sequence encoding saidendotoxin, and wherein the preselected DNA sequence further comprises aselectable marker gene or a reporter gene.
 98. The transgenic Zea maysplant of claim 90 wherein the selectable marker gene confers resistanceor tolerance to a compound selected from the group consisting ofhygromycin, sethoxydim, haloxyfop, glyphosate, methotrexate,imidazoline, sulfonylurea, triazolopyrimidine, s-triazine, bromoxynil,phosphinothricin, kanamycin, G418, 2,2-dichloropropionic acid andneomycin.
 99. The transgenic plant of claim 98 wherein the compound isphosphinothricin.
 100. The transgenic plant of claim 98 wherein thecompound is glyphosate.
 101. The transgenic plant of claim 98 whereinthe compound is kanamycin.
 102. The transgenic plant of claim 98 whereinthe compound is hygromycin.
 103. The transgenic plant of claim 90wherein the DNA encoding the Bacillus thuringiensis endotoxin is fusedin frame with said selectable marker or reporter gene.
 104. The inbredtransgenic plant of claim 96 wherein the DNA encodes a truncatedBacillus thuringiensis endotoxin.
 105. The hybrid transgenic plant ofclaim 99 wherein the DNA encodes a truncated Bacillus thuringiensisendotoxin.
 106. The transgenic plant of claim 86, 104 or 105 wherein thetruncated Bacillus thuringiensis endotoxin comprises about theN-terminal 50% of the endotoxin.
 107. The transgenic plant of claim 82or 90 wherein the preselected DNA further encodes a protease inhibitor.108. The transgenic plant of claim 87 or 95 wherein the preselected DNAsequence further comprises the maize AdhIS first intron or the maizeShrunken-2 first intron positioned between the promoter and the DNAencoding said endotoxin.
 109. The transgenic plant of claim 87 or 95wherein the preselected DNA sequence further comprises a manopinesynthase promoter, a nopaline synthase promoter or an octopine synthasepromoter.
 110. The transgenic plant of claim 87 or 95 wherein thepromoter is the CaMV 35S or 19S promoter.
 111. A population of plantsobtained by breeding the transgenic plants of claim 82 or 95 wherein thepreselected DNA sequence is transmitted by Mendelian inheritance throughboth male and female parent plants.
 112. An inbred insect-resistanttransgenic Zea mays plant prepared by a process comprising: (a) crossinga fertile transgenic Zea mays plant comprising a preselected DNAsequence encoding a Bacillus thuringiensis endotoxin, wherein thepreselected DNA sequence is adjusted to be more efficiently expressed inmaize than the B. thuringiensis DNA sequence encoding said endotoxin,and wherein said DNA sequence is heritable, with a member of a secondinbred Zea mays line; (b) recovering insect-resistant transgenic progenyplants from said cross; (c) back-crossing one of the transgenic progenyplants with a member of said second inbred line; (d) recoveringinsect-resistant transgenic progeny plants from said cross; and (e)repeating steps (b) and (c) to obtain said inbred plant.
 113. The inbredtransgenic Zea mays plant of claim 112 wherein said preselected DNAsequence encodes a truncated Bacillus thuringiensis endotoxin.
 114. Atransgenic insect-resistant hybrid plant prepared by crossing the inbredplant of claim 112 or 113 with an inbred Zea mays line, and recoveringsaid hybrid plant.